The immune mechanisms that provide protection against disease and death due to malaria remain poorly understood. Furthermore, the immune responses that contribute to protective immunity elicited by active vaccination in animal models of malaria are also poorly defined. Although elucidation of the exact mechanism(s) of immunity is not a prerequisite to the development of efficacious vaccines, in the absence of reliable in vitro correlates of protective immunity and with the lack of some understanding of what constitutes clinical immunity, development of effective malaria vaccines will continue to be empiric and haphazard at best. The major goals of this project are to clearly define the basis of protective immunity that develops after vaccination with subunit asexual blood stage vaccines in experimental animal models and develop relevant biological assays that will assist in the evaluation of candidate asexual blood stage malaria vaccines in humans. In addition, in the absence of either of the above, we are also taking an empiric approach to human vaccine development in the Aotus-P. falciparum monkey model. We have now established that protective immunity can be elicited routinely by subunit vaccination with the merozoite Major Surface Protein 1 (MSP1) in both murine (Plasmodium yoelii in mice) and primate (P. falciparum in Aotus monkeys) malaria models. In the P. yoelii murine model several lines of evidence (e.g., passive transfer of antibodies or T-cells, vaccination of gamma-chain, IL-12, IFN-gamma and IL-4 knockout mice and studies of congenic mouse strains) consistently indicate that protective immunity is antibody-dependent; however, immunogen-specific polyclonal antibodies alone, as assayed by total IgG and isotype-specific Ig ELISA, do not correlate with protective immunity. Although preliminary evidence suggested that the avidity and fine specificity of the polyclonal response contributed to protective immunity, the assays are not reliable enough to be surrogate markers of protective immunity. A variety of in vitro assays and vaccination studies in genetically or pharmacologically manipulated mice have been undertaken to determine the immune mechanisms that contribute to antibody-dependent protection. The data are consistent with a requirement for high antibody titers but not necessarily a required Th1 or Th2 response. The dependency on Freund' Adjuvant remains perplexing. The studies in primates, in which the use of Freund's adjuvant appears to be essential, have provided a basis for submission of an Investigational New Drug (IND) for testing of a MSP1-based subunit vaccine in Phase I human clinical trials. The completion of our first human Phase I trial with alum-adsorbed MSP1 emphasized the need for powerful adjuvants for immunogenicity. The role of Freund's adjuvant (which contains mycobacteria) is currently being investigated in studies of recombinant BCG. Studies of alternative delivery systems/regimens, for example, priming with naked DNA injections and boosting with conventional sub-unit vaccination, is being studied in primates to determine if a formulation other than Freund's adjuvant can elicit protective immunity in primates.